Regulation of B-lymphocyte proliferative responses by arachidonate metabolites: Effects on membrane-directed versus intracellular activators

twspmes try acachk)ionMe me4dMtes: EWects on membf8ne-dirscted versus intracettuliar activators Michael

G. Goodman,

M.D.,* and William

0. Weigle,

Ph.D. Lcr Jolla,

Cakf.

immunoregulator) #cts of the oxidative metaholites 01 arachidonic acid (AA) on prokferation i!f B-Lymphocytes were a.ssus.~ed in a serum-jree culture vy.stenz. Activation q/ H cells bj men&rune-directed 1igand.s and intraceltuiar actirutors \taaJ regulated by AA metabolites in ver? distinct.fashio,z,s. Thus exogenous cyclooxygenase product.r (particularly prostagiandins E, and E’,i_, amplified the response lo clnti-immunoRlobulin antihodie.\, whereas lipoqgenase products damped this response. in contrast. U cell activation bi+th K-mzrc,c~ptoKuclnosine (an intracellular cctivutorj was inhibited by cyiooxygenase prod1rct.s cmd remained relative!\ unaffected bj several Iipoxygenase products tested. This patt-ern of results was confirmed in studies with pathway inhibitors. Moreover, when liberation r/t ettdogenous AA W.LJ induced by .stimulativn oj phospholipase AL activity with melittin, inhibition of‘ the respnnjr to each class of activator was counteracted with the appropriate pathway inhibitor. Kesulth suggest that the two major groups of AA oxidation products function as a system ol~r‘ounterbakancin~ regulator?: irtfluences, serving to modulate R cell activation at the plusmu membrane and io downregulate B cell activation at the intracellular IcveI. 1.1AL~.ERGY CI.LV IMUMCJMI. 74:1/ 8. I%W, :

The event responsible for initiation of lymphocyte activation appears to be the interaction of informationbearing molecules with signal transducing structures located in the plasma membrane. Such interactions arc believed to perturb the receptor so that new asxociations between intramembranous proteins are formed. ultimatc,ly leading to signal transduction across the lipid bilayer. Recently, the use of purified anti-Ig has received considerable attention as a polyclonal analog for antigen, based on the fact that it interacts with immunoglobulin receptors for antigen 4 virtually all B cells. Much work has focused on cellular parameters of the B cell response to anti-l@.

!-xxr~ the Department of Immanotogy, Scripps Clinic and Research Foundation, La Jolla, Calif. ‘I’hts 1s publication No. 3263-IMM from the Department of im-rnunolgy. Scripps Clinic and Research Foundation, La Jolla, ( alif. Supported by United States Public Health Service Grants “1115284 and AI07Q07, Biomedical Research Support Grant RR@55 14. and American Cancer Society Grant 1M-42K. Reprint requests: Michael G Goodman. M.D., Department of Imnmnology. Scripps Clinic and Research Foundation. IO666 Y correy Pines Rd., La Jolla, CA 42007. *Recipient of United States Public Health Serwce Research Career %velnpment .4ward AI0017J 418

ilbbrevcations

8BrGuo: AA:

LPS: NDGA: HETE: TdK: HPETE:

usrd

Anti-immunoglobulin antibody 843romoguanosine %Mercaptoguanosine Arachidonic acid Lipopolysaccharide Nordihydroguaiaretic acid Hydroxy-eicosatetraenoicacid Thymidinc Hydroperoxy-eicosatetraenoicacid

FCS.

Fetal calf serum

PPD:

Purified protein derivative

fn recent work from this laboratory, we have described the striking immunobiologic activities of another class of I3 cell activator, the CS-substituted guanine ribonucleosides exemplified by 8Bfiuo and 8MGuo. This group of nucleosides is unique among immunostimulatory agents in that it consists of very low molecular weight analogs of one of the fundamental building blocks of cellular nucleic acid. The observation that these guanosine derivatives are mirogenic for murine splenic lymphocytes without the

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participation of cyclic guanosine monophosphate’.* wasfollowed by description of their capacity to induce polyclonal synthesisand secretionof immunoglobulin in both muTine and more recently human4lymphocytes. Uptake of 8BrGuo was found to proceed by carrier-mediated transport.5 Like that described for adenosine,the uptake system for 8BrGuo consists of high- and low-affinity components. Several lines of evidencesuggestthat this class of nucleosidetriggers the cell at an intracellular site. First, immobilization of thesecompoundsby covalent interaction involving either the guanine or ribose moieties of the nucleoside limits their accessto the interior of the cell andentirely abrogatestheir stimulatory activity. Second, B cell activation by 8MGuo and 8BrGuo is inhibited by nucleoside transport inhibitors at concentrationsthree to six orders of magnitude less than the optimal mitogenie concentrations of 8MGuo or 8BrGuo. Third, maneuversthat interfere with cell membranefunction (i.e., membrane rigidification or cross-linking of membraneimmunoglobulin profoundly interfere with stimulation by membrane-directedmitogens but not by carbon 8-substituted guanine ribonucleosides. The spleen cell population in which much of this work has been performed contains macrophagesas well as B- and T-lymphocytes. The macrophagecontains a high percentageof AA-esterified phospholipids in its plasma membrane, and the ability of this cell type to metabolize AA through cyclooxygenase and lipoxygenase pathways has been well studied.6-9For these reasons, we becameinterested in exploring the manner in which proliferative responsesof splenic B cells are regulatedby AA metabolites, with particular attention to their relative effects on membrane-directed versus intracellular activators. MATERIAL AND METHODS Mice. CBA/Cal male mice 8 to 16 weeks old were purchased from the Jackson Laboratory (Bar Harbor, Maine). Mice were maintained on Wayne Lab-Blox F6 pellets (Allied Mills, Inc.; Chicago, Ill.) and chlorinated water acidified with HCl to a pH of 3.0. Mitogens. Bacterial LPS 055:B5 was purchased from Difco Laboratories (Detroit, Mich.). F(ab’), fragments of goat antimouse p (anti-Ig) were obtained from Cappel Laboratories (Westchester, Pa.). 8MGuo was obtained from the Sigma Chemical Co (St. Louis, MO.). AA metabolites, inhibitors, and enzymes. AA, PGE,, PGE,, and PGF,,, prostacyclin, thromboxane BZ, indomethacin, NDGA, melittin, and soybean lipoxidase were obtained from the Sigma Chemical Co. Leukotriene D4 and 5-HETE were purchased from Calbiochem Behring (La Jolla, Calif.). Purity of all reagents was verified by high-pressure liquid chromatographic analysis (>97%). Lymphocyte cultures. Spleen cell suspensions were pre-

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419

25-

i

..4x1$&o.’ &

2xb”

5xb

Concentration of NOGA [Ml

FIG. 1. Viable CBAlCaJ B cells (4 x IO51were cultured with BMGuo (ImM) or anti-19 (50 kg/ml) in the presence of incremental concentrations of NDGA. Cultures were labeled with [3H]TdR for the final 24 hours of incubation. Results are presented as percent [3HITdR uptake in cultures containing no NDGA.

pared as described previously.‘” Unless specifically noted, cells were cultured in serum-free medium, whose constituents have been detailed elsewhere,“’ in microculture plates (No. 3546 Costar; Cambridge, Mass.) at 4 X lo6 viable cells/ml in 0.1 ml. Microcultures were incubated at 37” C in a humidified atmosphere of 5% CO2 in air. Preparation of T cell-depleted populations. B cell-enriched populations were prepared by intraoperitoneally injecting mice with 60 ~1 of rabbit antimouse undiluted antithymocyte serum (Microbiological Associates; Bethesda, Md.) 2 days before sacrifice.” Spleen cell populations generated from these animals were then further treated with 1:lOOO monoclonal anti-Thy 1.2 (New England Nuclear; Boston, Mass.) for 30 minutes at 4” C. Treated cells were centrifuged at 280 X g for 10 minutes, antibodies were removed, and cells were resuspended in a 1:6 dilution of CBA red blood cell-absorbed guinea pig complement at 37” C for 45 minutes. Cells were then washed and cultured as above. Resulting populations were at least 99% free of T cells as measured by immunofluorescence. Measurement of [WjTdR uptake. To evaluate deoxyribonucleic acid synthesis, cells were radiolabeled with 1.O pCi [3H]TdR per culture (5 Ci/pM, Amersham Radio Chemicals; Amersham, England) during the final 24 hours

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Concentration of lmidazole !Mi FIG. 2. Viable CBAiCaJ spleen cells (4 * IOYwere cultured with 8MGuo or anti-lg in the presence of incremental concentrations of imidazole. Cultures were labeled with i’HjTdR for the final 24 hours of incubation. Results are expressed as the arithmetic mean (-r SE) of five replicate zultures

of culture. Microcultures were harvested with a Brandel 3Aodei M24V cell harvester (Biological Research and Development Laboratories; Rockville, Md. ! onto glass fibe: filter strips. Filter disks were transferred to plastic scintll iation vials, covered with liquid scintillation cocktail, and counted in ;I Beckman LS-7500 liquid scintillation specrwmeter,

RESUtTS &fl;?ct of fipoxygertase inhibition on B cell activation

hv unfi-/g OY&V&o. Effects of incremental concen[rations of the lipoxygenase inhibitor NDGA were assessedon cultures of splenic B cells activated with optimal mitogenic concentrations of 8MGuo ( 1mM) tar anti-Ig (SO p,g/ml). These two B cell activators exhibit very different responses to the inhibitor: XMGuo-activatedcells exhibit a linear pattern of inhibition, whereas the effect of anti-Ig stimulation is biphasic, with initial enhancementfollowed by inhibition (Fig. I). BecauseNDGA also inhibits thromboxanesynthetase,the effect of inhibiting this enzyme was assesseddirectly. Addition of increasing concentrations of either imidazole (or benzylimidazole; data not shown) to culture failed to inhibit the responseto either 8MGuo or anti-& (Fig. 2). Effect of cyclooxygenase inhibitiotz on B cell ucti-

~clrionhv anti-Zgor 8,VGuo. Increasingconcentrations

a

10’5 2x10’”

I

I

I

3x10’5 4x10’5

Concentration of lndomethacin [Ml

FIG. 3. Viable Cl3AiCaJ spleen ceils (4 x 107 were cultured with 8MGuo or anti-19 in the presence of incremental concentrations of indomethacin. Conditions of labeling and expression of results are as in Fig. 1.

of indomethacin were added to cultures of CBA/Cal spleen cells activated with either 8MGuo or anti-lg. Again. very divergent effects were exerted on cells activated by membrane-directedversus intracellular activators (Fig. .!). Thus the response to 8MGuo showed modestenhancementby exposureto concentrations of 3OpM to 4(.&M, whereasresponseto antiIg was approximately 50% inhibited in this dose range. The samepattern was observed when acetylsalicylic acid was used to inhibit the cyclooxygenase pathway. Abilic qf endogenous AA to modulate proliferative responses tc?nnti-lg or 8MGuo. When spleen cells

activated with 8MGuo or anti-Ig were cultured with increasingconcentrationsof AA, no significant effect could be observed up to 1OkM (data not shown). To stimulate liberation of endogenous AA from phospholipids. incremental concentrations of melittin, a known stimulant of phospholipaseA2, were addedto cultures activated with either 8MGuo or anti-lg. Proliferative responseto each activator was inhibited by melittin in a dose-dependentfashion (Fig. 4). When 1p,M NDGA was added to cultures stimulated with anti-Ig, melittin-induced inhibition was markedly alleviated. Moreover, when 8kM indomethacin was

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metabolites

0 O

.Ol

0.1

1.0

0 Concentration

of Melittln

.Ol

0.1

1.0

Ipglmll

FIG. 4. Viable CBAiCaJ spleen cells (4 x 105) were cultured with anti-19 in the presence or absence of IpM NDGA with incremental concentrations of melittin (left). Identical cultures activated with 8MGuo in the presence or absence of 8f.~M indomethacin were cultured with incremental concentrations of melittin (right). Conditions of labeling and expression of data are as in Fig. 2.

addedto cultures stimulated with 8MGuo, the inhibitory effect of melittin was again significantly reduced. Here it appearsthat endogenously processedAA is converted to PGs, with a dominant inhibitory effect relative to any positive effect of lipoxygenase products. The converse of these experiments is shown in Fig. 5, where the inhibitory effect of melittin on antiIg is exaggeratedby including 8pM indomethacin and the inhibitory effect on 8MGuo is enhanced in the presenceof 1pM NDGA. Effect of lipoxygenaseproducts on B cell activation by anti-Zg or 8MGuo. Becausethe results of the preceding experiments could be accountedfor either by blocking the effect of an end product or by increasing an opposite effect attributable to channeling excess substrate into the unblocked pathway, experiments were undertakenin which end products of each pathway were added directly to B cells undergoing stimulation with either anti-Ig or 8MGuo. Over a concentration range of lo-‘*M to 10m6M,5-HETE had no significant modulatory effect on responseto 8MGuo (Fig. 6). When its effects on anti-Ig-induced stimulation were assessed,however, a dose-dependentinhibitory effect was observed, with a plateauat lo-“M. Effects of 15-HPETE or 15-HETE generated by treatment of AA with soybeanlipoxidase were evaluated next (Fig. 7). This preparation has been characterized previously. ‘* Again, activation with the membrane-directedligand wasconsiderablymore sensitive to inhibition by this agent than was activation by 8MGuo. Leukotriene D, over a concentrationrange of 10-13M to lo-‘M failed to modulate responsesto either anti-Ig or 8MGuo (data not shown).

0

.Ol

0.1

1.0

0

.Ol

0.1

1.0

Concentrationof Melittin (pglmll FIG. 5. Viable CBA/CaJ spleen cells (4 x 105) were cultured with anti-19 with or without indomethacin (8pM) in the presence of incremental concentrations of melittin ((Iefr). Identical cultures were activated with 8MGuo in the presence or absence of lf.~M NDGA and incremental concentrations of melittin (right). Conditions of labeling and expression of data are as in Fig. 2.

Effect of cyclooxygenaseproducts on B cell activation by anti-Q or 8MGuo. In studiescomplementary to those in the preceding section, incremental concentrations of PGE, were addedto cultures of spleen cells activated with either anti-Ig or 8MGuo. Whereas responseto 8MGuo was inhibited in a dose-dependent ponseto anti-Ig was strongly enhancedin to the concentrationadded(Fig. 8). In light results published by others examining the PGs on T cell activation by lectins, the effect of PGs on B cell stimulation by a directed ligand was unexpected. Because

422

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FIG. 6. Viable CBAXaJ spleen cells (4 + IO”) were acti. vated with anti-lg or 8MGuo in the presence of incremental concentrations of 5-HETE. Conditions of labeling and expression of data are as in Fig. I

CLIN. IMMUNOL SEPTEMBER 1984

FIG. 8. Viable CBAKaJ spleen cells (4 x IO51were cultured with anti-19 or 8MGuo in the presence of incremental concentrations of PGE,. Conditions of labeling and expression of data are as in Fig. 2.

109A

Activator "--__-

PGL

8MGuo EMGuo

+

Anti-lg Am4g

”

PPO

Ant,.lg

PPD

+ I ny---3----

*I

I 10"

10'

I

10'

I

10'

I 1!I"

Concantralmn01 IWPETE IMi

FIG. 7. Viable CBAXaJ spleen cells (4 x 105) were cultured with anti-lg or 8MGuo in the presence of incremental concentrations of 15-HPETE. Conditions of labeling and exoression of data are as in Fig. 1

these other systems all depend on the presence of FCS. w examined the effects of PGs on anti-lp stimulation In the presence of FCS. At lOp,M PGE,. a concentration that doubled the magnitude of the response to antl-lg in the absence of serum, this response was inhibited approximately 50% in the presence of serum (Fig. 9). PPD. another surface-directed mitogen. and XMGuo were also inhibited

,

20 30 40 IlHITdR Uptake lcpm x 10%ulturel

I

50

FIG. 9. Viable CBAiCaJ spleen cells (4 x 105J were activated with 8MGuo (ImM), anti-19 (50 pgimt), or PPD (300 pgirnl) in the presence or absence of IO’5 PGE,. All cells were cultured in the presence of 5% FCS-containing medium. Conditions of labeling and expression of data are as in Fig. 2

Hecause many reports have indicated that PGs indirectly exert their effect5 on lymphocyte proliferative responses through suppressor ‘I‘ cells.” ” effects of a number of PGs on 1‘ cell-depleted spleen cell populations were evaluated. Under these circumstances. the earlier dichotomy was again observed in the absence of FCS (Fig. IO). PGE, and PGE, had similar effects. with PGE2 being slightly more effective. Interestingly. PGF!, fully retained its enhancing activity for the response to anti-lg while retaining very little Inhibitory activity for the response to 8MGuo. The

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Concentration of PGEI [Ml

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423

Concentration of PGE2[Ml

yv Concentration of PGFzo [Ml

by arachidonate

10”

10.6

10-s

Concentration of PGI, [Ml

FIG. 10. Viable CBA/CaJ B cells (4 x 105) were activated with anti-19 or 8MGuo in the presence of incremental concentrations of PGE, (upper left), PGE, (upper right), PGFSu (lower IeftJ, or PGI, (lower right). Conditions of labeling and expression of data are as in Fig. 2.

enhancing activity of exogenousprostacyclin (PGI,) for the responseto anti-Ig was considerably diminished and it had no discernableeffect on the response to SMGuo. As a control, exogenous thromboxane B2 had no effect on the responseto anti-Ig and a minimal inhibitory effect on the responseto 8MGuo (data not shown). B cell subpopulations responsive to 8MGuo and anti-lg. The diametrically opposite effects of AA ox-

idation products on responsesto membrane-directed and intracellular activators posesan interesting question: Do both agentsact on a single subpopulation of B cells such that the opposing effects occur within that group of B cells, or are these effects exercised in distinct B cell subpopulations?Additivity experimentswere carried out on B cell-enriched populations with optimal stimulatory concentrations of each activator that wasevaluated(Fig. 11). Thesedataclearly indicated that the group of B cells activated by antiIg is distinct from the group stimulated by LPS, 8MGuo, or 8BrGuo. The latter two agents appearto

stimulate essentially the same subpopulation, which appearsto be a subsetof that activated by LPS. DISCUSSION Data presentedin this article suggestthat the different oxidation productsof AA function asa balanced regulatory system modulating lymphocyte activation. This sytem of counterbalancing regulatory activities is clearly illustrated in the caseof membrane-directed activators. B cell activation by either anti-Ig antibodies or by LPS (data not shown) is subject to inhibitory signals provided by lipoxygenase pathway products. Currently, it appearsthat the HETEs and/ or HPETEs particularly exhibit this activity. The sole leukotriene testedto date, LTD4, has failed to exhibit significant biologic activity in this system.Evaluation of other leukotrienes and HETEs is planned. B cell activation by membrane-directedactivators such as anti-Ig is subject to amplification by cyclooxygenasepathway products. PGE, appearsto be active at lower concentrations than either PGE, or PGF2,.

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In contrast to that by membrane-directed mitogens, B-lymphocyte activation by 8MGuo is inhibited by products of rhe cyclooxygenase pathway. PGE, and PGE: appear to have roughly comparable inhibitory activity, whereas PGF2, has only marginal activity and prostacyclin and thromboxane B, lack significant activity altogether. The existence of such a hierarchy suggests that B cell receptors for these molecules are of meticulous chemical specificity, the difference between a carbonyl and 3 hydroxyl functionality mandating significant differences in elicited biologic activity.

HE. 11. Viable CBA/CaJ B celts (4 i. 107 were cultured in the presence of optimal concentrations of anti-lg. 8MGuo, 8BrGuo. or LPS. Certain of these cultures were activated with optimal concentrations of two mitogens as shown. Results are presented as the arithmetic mean counts per minute of five replicate cultures. The diagram represents the relationship between B cell subsets as suggested by tabuiar data.

Prostacyclin exhibits considerably less activity than of the above three compounds. and thromboxane B-, is entirely devoid of such activity. Results of studies with various enzyme inhibitors were entirely consistent with the pattern described above. Thus inhibition of PC synthetase with either mdomethacin or acetylsalicylic acid diminished the zlagnitude of the response to anti-Ig, whereas exposure to low concentrations of the lipoxygenase inhibitor NDGA enhanced this response. Effects of indo-.methacin are most likely not a result of inhibition o1 conversiori of HPETE to HETE,‘” since 5-HETE had the same effect as indomethacin. Inhibitory effects oi higher doses of NDGA appeared to involve loss of’ cellular viability. Because this pattern was so diametrically opposed to that observed for activation with 8MGuo. it suggests that if membrane activation results in the generation of an intracellular messenger of the carbon--substituted guanine ribonucleoside species. stimulatory effects of PGs operating at the plasma membrane must ovemide the inhibitory effects exerted on such messengers within the cell. Moreover. when generation of AA from membrane phospholipids was stimulated by the action of melittin on phospholipase A,. inhibition of the response to anti-lg. presumably by lipoxygenase products, was ameliorated by block.ing this pathway with NDGA. Regulation at the level of intracellular activation appears to operate in a sotnewhat different fashion. an!;

None of the products of the lipoxygenase pathway that were tested (with the possible exception of lSHPE,TE) appears to have any significant modulatory activity for B cell stimulation by SMGuo. That this pathway can be downregulated by PGs suggests that, unless other lipoxygenase products prove to stimulate [his response. there has heen no selective advantage to amplifying the response at this level during evolution of this system. Results of studies with inhibitors of the oxidative pathways of AA metabolism are conhistent with the above conclusions. Thus blockade of the cyclooxygenase pathway with indomethacin failed to inhibit the response to SMGuo, whereas this rehponse was extremely sensitive to inhibition by the lipoxygenase inhibitor NDGA. Moreover, when generation of endogenous AA was stimulated with melittin. inhibition of the response to SMGuo, presumably by production of PGs, could be significantly alleviated by blocking this pathway with indomethacin. In contrast, blockade of the lipoxygenase pathway with NDGA accentuated inhibition. REFERENCES i Goodman M. Weigle W: Activation of lymphocytes by brommated nucleoside and cyclic nucleotide analogues: Implications fbr the “second messenger” function of cyctic GMP. Proc Nat1 Acad Sci USA 78:7604. 1981 2 Goodman M, Weigie W: Bromination of guanosine and cyclic GMP confers resistance to metabolic processing by B cells. .I lmmunol !29:2715. 1982 : Goodman M. Weigle W: Induction of immunoglobulin secre[Ion by a \lmple nucleoside derivative. J Immunol I28:2399. I982 -1. Osundwa V, Ledgley C, Martin C, Dosch H: Activation of human B lymphocytes by X-bromoguanosine (8’BG). Fed Proc 4’:409. 198.3 4 Goodman M, Weigle W: Intracellular lymphocyte activation and carrier-mediated transport of C&substituted guanine nbonucleobide. Proc Nat1 Acad Sci USA 81:862, 1984 h. Rouzer C. Scott W. Hamill A, Liu F-T, Katz D, Cohn 2: Secretion of leukotriene C and other arachidonic acid metabelites by macrophages challenged with immunoglobulin E immune complexes. J Exp Med 156: 1077, 1982 7. Bach M. Brashler J, Hammarstriim S. Samuelsson B: Identi-

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fication of leukotriene C-l as a major component of slowreacting substance from rat mononuclear cells. J Immunol 125:115, 1980

8. Rankin J, Hitchcock M, Merrill W, Bach M, Brashler J, AskenaseP: IgE-dependentreleaseof leukotrieneC, from alveolar macrophages.Nature 297:329, 1982 9. Hsueh W, Desai U, Gonzalez-CrussiF, Lamb R, Chu A: Two phospholipase pools for prostaglandin synthesis in macrophages. Nature 290:710, 1981 10. Goodman M. Fidler J, Weigle W: Nonspecific activation of murine lymphocytes. IV. Proliferation of a distinct, latc-maturing lymphocyte subpopulation induced by 2-mercaptoethanal. J immunol 121:1905. 1978 11. Harwell L, Skidmore B, Marrack P, Kappler J: Concanavalin A-inducible, interleukin-2-producing T cell hybridoma. J Exp Med 152:893, 1980 12. GoodmanM, Weigle W: Modulation of lymphocyte activation. I. Inhibition by an oxidation product of arachidonic acid. J Immunol 125:593, 1980 13. Goodwin J, Bankhurst A, Messner R: Suppressionof human T-cell mitogenesis by prostaglandin. J Exp Med 146:1719, 1977 14 Webb D, Nowowiejski I: Mitogen-induced changes in lymphocyte prostaglandinlevels: a signal for the induction of suppressorcell activity. Cell Immunol 41:72, 1978 15 Fulton A, Levy J: The induction of nonspecificT suppressor lymphocytes by prostaglandin E,. Cell Immunol 59:54, 1981 16. Siegel M, McConnell R, Porter N, CuatrcasasP: Arachidonate

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metabolismvia lipoxygenase and I&hydroperoxy-5,8,10,14icosatetraenoicacid peroxidase sensitive to anti-inflammatory drugs. Proc Nat1 Acad Sci USA 77:308, 1980

DISCUSSION Charles Parker: Were the experiments with thromboxane B, done in serum? If they were, rhe serum itself would have substantial amounts of thromboxane B? in it. Michael Goodman: We had no serum in the system. Marc Goldyne: Do you have any idea as to whether the effect of .5-HETEon anti-Ig activation of B cells is specific, or will other hydroxy acids produce the same results? M. Goodman: I would like to know more about the specificity of the 5-HETE effect. Jack Vanderhuek: Just a comment. We have looked very hard to find any metabolites of AA produced by B cells in Balb/C mice and were not able to find any products. In contrast, T cells did convert AA to diverse products. Aaron Marcus: It would be of interest to know the profile of AA metabolism in the cells you are studying. M. Goodman: Because of the presence and contributions of macrophages to B cell function, this will be a complex series of experiments. However, the results will be important in providing a perspective for the interpretation OFthe effects of exogenoussynthetic metabolites of AA.